Electron-Transfer Reactivity of Metalloproteins in Folded, Partially Unfolded, and Completely Unfolded Forms Scott Ichm Ael Tremain Iowa State University
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2002 Electron-transfer reactivity of metalloproteins in folded, partially unfolded, and completely unfolded forms Scott ichM ael Tremain Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Inorganic Chemistry Commons Recommended Citation Tremain, Scott ichM ael, "Electron-transfer reactivity of metalloproteins in folded, partially unfolded, and completely unfolded forms " (2002). Retrospective Theses and Dissertations. 487. https://lib.dr.iastate.edu/rtd/487 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Electron-transfer reactivity of metalloproteins in folded, partially unfolded, and completely unfolded forms by Scott Michael Tremain A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Inorganic Chemistry (Biomolecular Science) Program of Study Committee: Nenad M. Kostic, Major Professor Parag R. Chitnis Victor S.-Y. Lin John G. Verkade L. Keith Woo Iowa State University Ames, Iowa 2002 UMI Number: 3061872 ® UMI UMI Microform 3061872 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Scott Michael Tremain has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major ProfeMor Signature was redacted for privacy. For the j r Program iii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION 1 General Overview 1 Dissertation Organization 3 References 4 CHAPTER 2. FATE OF THE EXCITED TRIPLET STATE OF ZINC CYTOCHROME C IN THE PRESENCE OF IRON(III), IRON(II), IRON-FREE, AND HEME-FREE FORMS OF CYTOCHROME C 6 Abstract 6 Introduction 7 Experimental 9 Chemicals 9 Buffers 9 Proteins 9 Kinetics 10 Calculations 11 Results and Discussion 12 Natural Decay of 3Zncyt 12 Effects of Possible Quenchers on Decay of 3Zncyt 13 Possible Mechanisms of Quenching 14 Absence of Enhanced Radiationless Decay 14 Presence of Energy Transfer 15 The Question of Reductive Quenching of 3Zncyt 17 Oxidative Quenching of 3Zncyt 18 Bimolecular Rate Constants 19 Kinetic Effects of Ionic Strength 20 Conclusions 20 iv Supplementary Material 21 Acknowledgements 21 References. 21 Tables and Figures 25 Supplementary Material 29 CHAPTER 3. MOLTEN-GLOBULE AND OTHER CONFORMATIONAL FORMS OF ZINC CYTOCHROME C. EFFECT OF PARTIAL AND COMPLETE UNFOLDING OF THE PROTEIN ON ITS ELECTRON-TRANSFER REACTIVITY 34 Abstract 34 Introduction 35 Experimental Section 39 Chemicals 39 Proteins 39 Preparation of Buffer and Dénaturant Solutions 40 Preparation of (Un)folded Forms of Zncyt 40 Circular Dichroism and Absorption Spectroscopy 41 Equilibrium Unfolding of Zncyt 41 Laser Flash Photolysis 42 Reaction Mechanism 44 Fittings of Kinetic Data 44 Results 45 Characterization of (Un)folded Forms of Zncyt by Absorption Spectroscopy 45 Equilibrium Unfolding of Zncyt 45 Characterization of (Un)folded Forms of Zncyt by Circular Dichroism Spectroscopy 45 Characterization of (Un)folded Forms of Zncyt by Natural Decay of the Triplet State 46 Conformational Transitions Between (Un)folded Forms of Zncyt 47 Demetalation of Zncyt and Remetalation of Hzcyt by Zinc(II) Ions 48 Effect of Zncyt Conformation on the Porphyrin Affinity for Zinc(II) Ions 49 V Oxidative Quenching of 3Zncyt by Inorganic Complexes and Cyt(HI) 49 Discussion 51 Structure and Reactivity of (Un)folded Forms of Cyt(III) and Zncyt 51 Absorption and Circular Dichroism Spectra of (Un)folded Forms of Zncyt 51 Natural Decay of the Triplet State of Zncyt in Different (Un)folded Forms 53 Reactivity of 3Zncyt in the Five (Un)folded Forms 54 (A) Role of Electrostatic Interactions 54 (B) Partial Unfolding of Zncyt: The Molten-Globule Form 55 (C) Complete Unfolding of Zncyt: The Uacid and Uurea Forms 56 Conclusions 57 Supplementary Material 57 Acknowledgements 57 References 57 Tables and Figures 64 Supporting Information 75 CHAPTER 4. GENERAL CONCLUSIONS 80 APPENDIX. OPTIMIZATION OF A BACTERIAL EXPRESSION SYSTEM FOR THE SOLUBLE FORM OF TURNIP CYTOCHROME F AND ITS ISOLATION 83 Introduction 83 Experimental 86 Expression 86 Purification 88 Results and Discussion 90 Expression 90 Purification 91 Conclusions 93 References 94 Tables and Figures ACKNOWLEDGMENTS 1 CHAPTER 1. GENERAL INTRODUCTION General Overview With the sequencing of the human genome complete, attention has shifted to studying the structure and stability of proteins, protein-protein and protein-ligand interactions, structure-function relationships, and how these relate to disease and drug discovery.1 The biological function and reactivity of a protein depends critically on its ability to adopt a specific and unique three-dimensional structure. When a protein is synthesized in the cell, its conformation approximates a random-coil, lacking most secondary and tertiary structure. Utilizing only its sequence of amino acids, proteins spontaneously fold into their native conformations under physiological conditions. A protein can efficiently fold from many unfolded states to one native state on the time scale of milliseconds to seconds. Understanding how this process occurs is one of the great challenges in science.2"5 Metal ions and organic moieties are essential components of proteins involved in biological processes, such as respiration, metabolism, and photosynthesis. Nearly one-third of all proteins require cofactors to bind and activate substrates, transfer atoms or groups, transfer electrons, and provide structure. A class of proteins utilizing a cofactor to perform its biological function is cytochrome c. C-type cytochromes are defined as electron transfer proteins which have an iron porphyrin (heme) covalently attached to the protein backbone through cysteine residues.6 Because of their biological roles and favorable chemical and spectroscopic properties, these proteins were used in many important biochemical and biophysical studies.7"8 Especially relevant are recent studies on the folding of horse heart 2 cytochrome c.9"11 One strategy that can provide insight into the mechanism of protein folding is to detect and characterize the intermediates in the folding pathway. One such intermediate is the molten globule, a compact denatured form with a significant amount of nativelike secondary structure, but largely flexible and disordered tertiary structure. In comparison with the native folded form, the internal hydrophobic core of the molten globule is more exposed to solvent and the side chains are more mobile.12 Characterization of these intermediates by traditional crystallographic or NMR methods is difficult because of the dynamic, heterogeneous, and often transient nature of these nonnative states. Our approach involves measuring the intrinsic chemical reactivity of proteins in various conformational forms. Cytochrome c is our protein of choice because its folding mechanism has been thoroughly investigated and the covalently-bound heme prosthetic group provides an excellent optical marker. Various conformational forms can be captured in solution by simply changing the conditions (pH, presence of anions, presence of dénaturants). At pH 2 in the presence of stabilizing anions, the molten-globule form of cytochrome c can be obtained. The replacement of redox-active heme iron by redox-inactive zinc(II) in cytochrome c, allows study of reactions between heme proteins, which otherwise have overlapping absorption spectra. The long-lived triplet state of the zinc porphyrin, designated 3Zncyt, is easily produced by laser flash and is a strong reductant. Moreover, we can study photoinduced electron transfer from 3Zncyt to an electron acceptor without the complications of external reductants. 3 As a prerequisite to studying the reactivity of proteins in partially and completely unfolded forms, we investigate photoinduced electron-transfer